1. Field of the Invention
The present invention relates to methods for producing copies of a nucleic acid and for detecting the presence of polynucleotide analytes. Particularly, the present invention relates to the prevention of amplification of a nucleic acid contaminant from one sample to the next during the above methods.
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the .sup.32 P labelling of DNA with T4 kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid or tissue and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
Recently, a method for the enzymatic amplification of specific double stranded sequences of DNA known as the polymerase chain reaction (PCR) has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the desired sequence flanked by the primers. The two different PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers.
Another method that has recently been described is an amplification of a single stranded polynucleotide using a single polynucleotide primer. The single stranded polynucleotide that is to be amplified contains two non-contiguous sequences that are complementary to one another and, thus, are capable of hybridizing together to form a stem-loop structure. This single stranded polynucleotide may be already part of a polynucleotide analyte or may be created as the result of the presence of a polynucleotide.
Another method for achieving the result of an amplification of nucleic acids is known as the ligase chain reaction (LCR). This method uses a ligase enzyme to join preformed nucleic acid probes. The probes hybridize with the nucleic acid analyte, if present, and ligase is employed to link the probes together resulting in two templates that can serve in the next cycle to reiterate the particular nucleic acid sequence.
Another method for achieving a nucleic acid amplification is the nucleic acid sequence based amplification (NASBA). This method is a primer-directed, enzymatic process that induces in vitro continuous, homogeneous and isothermal amplification of a specific nucleic acid.
Another method for amplifying nucleic acids is the Q-beta-replicase method, which relies on the ability of Q-beta-replicase to amplify a specific RNA substrate exponentially and is used as a label to detect binding rather than a method to create more target nucleic acid.
One method for detecting nucleic acids is to employ nucleic acid probes. One method utilizing such probes is described in U.S. Pat. No. 4,868,104, the disclosure of which is incorporated herein by reference. A nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support.
Detection of signal depends upon the nature of the label or reporter group. If the label or reporter group is an enzyme, additional members of the signal producing system would include enzyme substrates and so forth. The product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electrometric means. If the label is a fluorescent molecule, the medium can be irradiated and the fluorescence determined. Where the label is a radioactive group, the medium can be counted to determine the radioactive count.
For any of the above methods for amplifying nucleic acid there is a risk of contaminating the amplification mixture with previously amplified material and thereby amplifying material that was not present in the original sample, namely, a contaminant. The quantities of amplification product can be very large thereby aggravating the potential contamination. Once aerosols of amplified nucleic acid are produced in a laboratory, droplets containing this material can invade subsequent amplification mixtures or equipment. Attempted amplification of a nucleic acid may then produce amplified copies of this contaminating material even when the target nucleic acid, or sequence thereof, was not present in the sample being amplified. Such contamination can also occur if the same container is employed for multiple amplifications even though the container is cleaned.
As few as one molecule will sometimes be sufficient to contaminate other containers that are to be used in further amplifications. This possibility for contamination can result in a false test since such a single molecule can be amplified and detected. The result of the test will not accurately reflect the presence or absence of the particular nucleic acid in the patient sample being tested.
Recently, a containment cuvette for amplification of nucleic acids has been disclosed. The cuvette and its method of use are designed to prevent amplified nucleic acid from being released into the atmosphere.
The need still exists for methods for carrying out assays that avoid false positives caused by cross-contamination of samples, are preferably homogeneous and are automatable with relatively simple instrumentation.
2. Description of the Related Art
U.S. Pat. No. 5,035,996 (Hartley) discloses a process for controlling contamination of nucleic acid amplification reactions. An amplification procedure is performed on a first sample in which one or more of the four normal ribonucleoside triphosphates or deoxyribonucleoside triphosphates is replaced with an exo-sample nucleotide. After amplification, any contaminating amplified product that may be remaining is subjected to a physical, chemical, enzymatic, or biological treatment that renders nucleic acid containing the exo-sample nucleotide substantially unamplifiable. The treatment may be done as a separate step or it may be done in the presence of a second sample containing nucleic acid sequences to be amplified. The amplified nucleic acid sequences derived from the first sample which contaminates the second sample are not further substantially amplified during amplification of nucleic acid sequences of the second sample.
International Patent Application No. PCT/US91/03052 discusses a method for reducing carryover contamination in an amplification procedure. The method involves incorporation of at least one modification into the amplification product. A modification is preferably incorporated into an amplification product by using presynthesized amplification probes or primers that contain the selected modification. The modified amplification product is readily distinguishable from the target sequence in a test sample. Prior to amplifying the target in a new test sample, the sample may be treated to selectively eliminate the contaminant amplification product so that it cannot be amplified in the new sample. The modifications may include the introduction of a ligand such as biotin or fluorescein into the contaminant amplification product. The resulting biotin- or fluorescein-modified amplification product can be removed from subsequent test samples by contacting these samples with immobilized avidin or anti-fluorescein antibody, respectively.
Longo, et al., in Gene (1990) 93:125-128, describe the use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. The method has two steps: (i) incorporating dUTP in all PCR products (by substituting dUTP for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UDF), followed by thermal inactivation of UDG. UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines. This method was applied by Pang, et al., in Molecular and Cellular Probes (1992) 6:251-156, for the control of contamination in the PCR-based amplification of RNA.
Walder, et al., in Nucleic Acids Research (1993) 21(18):4339-4343, discuss the use of PCR primers containing a 3'-terminal ribose residue to prevent cross-contamination of amplified sequences.
Two alternative protocols for pre-PCR sterilization which utilize exonuclease III, which catalyzes the sequential cleavage of 5'-mononucleotides from the 3'-hydroxyl end of duplex DNA, are described by Zhu, et al., in Nucleic Acids Research (1991) 19(9):2511.
The use of geometric differences allowing for differential enzymatic inactivation of PCR product and genomic targets in preventing carry-over contamination in PCR is discussed by Muralidhar, et al., Gene (1992) 117:107-112.
Cimino, et al., in Nucleic Acids Research (1991) 19(1):99-107, disclose a post-PCR sterilization method to control carryover contamination for the polymerase chain reaction. See also Issacs, et al., Nucleic Acids Research (1991) 19(1):109-116.
The use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents is described by Meier, et al., in Journal of Clinical Microbiology (1993) 31(3):646-652. The use of ultraviolet light alone to eliminate sources of contamination in PCR is discussed by Sarkar, et al., in Nature (1990) 346:27.
Furrer, et al., in Nature (1990) 343:324, describe treatment of individual reaction mixtures in PCR, before adding template DNA and Taq polymerase, with DNaseI or restriction endonucleases that cut internal to the pair of amplification primers to prevent amplification of contaminating DNA.
PCT Patent Application No. PCT/FR91/00513 (Brandys, et al.) discloses an improvement to the method of in vitro enzymatic amplification (PCR method) of a target DNA sequence present in heterologous DNA in a medium that comprises a DNA polymerase and a primer oligonucleotide. The method includes various cyclically repeated amplification steps whereby, at a chosen time following the end of the amplification cycles, the resulting amplification products are made unsuitable for later reamplification and/or before the start of the PCR reaction, a pretreatment is carried out to selectively prevent any reamplification of the previous amplification products resulting from a PCR reaction using the same primer oligonucleotides.
Corey, et al., in Biochemistry (1989) 28(21):8277-8286 disclose the generation of a catalytic sequence-specific hybrid DNase.
Biotinylated dUTP is discussed by Lo, et al., "PCR Protocols: A Guide to Methods and Applications" (1990) Academic Press, San Diego, Calif..
A containment cuvette for conducting PCR is disclosed in European Patent Application publication number 0 381 501 (Schnipelsky, et al.). Detection reagents are either pre-incorporated into compartments in the cuvette or are added after amplification. In the latter situation a check valve prevents amplified nucleic acid from being released. Transfer of liquids between compartments is achieved by the use of flexible compartment walls and an external pressure source or by pistons that are part of the cuvette and operate on the compartments as a piston within a piston chamber.
A device for processing biological specimens for analysis of nucleic acids is described in U.S. Pat. No. 5,188,963. The device has a hinged compartment facilitating automation of DNA- and RNA-based diagnostics and genetic surveillance and detection. Specimens are embedded in a matrix in the carrier. The matrix is then treated by one or more of the techniques such as amplification, electrophoresis, and hybridization as selected for the desired analysis and then the sample is treated to detect the cellular component.
A process for amplifying, detecting and/or cloning nucleic acid sequences otherwise referred to as PCR is disclosed in U.S. Pat. Nos. 5,008,182, 4,965,188, 4,800,159, 4,683,195 and 4,683,202. Sequence polymerization by PCR is described by Saiki, et al., (1986) Science, 230: 1350-1354.
U.S. patent application Ser. Nos. 07/299,282, abandoned and 07/399,795, abandoned, filed Jan. 19, 1989, and Aug. 29, 1989, respectively, describe nucleic acid amplification using a single polynucleotide primer. U.S. patent application Ser. No. 07/555,323 filed Jul. 19, 1990, discloses methods for producing a polynucleotide for use in single primer amplification. U.S. patent application Ser. No. 07/555,968 filed Jul. 19, 1990 U.S. Pat. No. 5,439,793, describes a method for producing a molecule containing an intramolecular base-pair structure. U.S. patent application Ser. No. 07/776,538 filed Oct. 11, 1991, abandoned, discloses methods for producing a polynucleotide for use in single primer amplification. U.S. patent application Ser. No. 07/923,079 filed Jul. 31, 1992 abandoned, describes a method for introducing sequences at the 3' end of polynucleotides. The disclosures of these six applications are incorporated herein by reference in their entirety.
Other methods of achieving the result of a nucleic acid amplification are described by Van Brunt in Bio/Technolgy (1990) 8(No.4): 291-294. These methods include ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and Q-beta-replicase amplification of RNA. LCR is also discussed in European Patent Applications Nos. 439,182 (Backman I) and 473,155 (Backman II).